SUMMARY Osteocyte function is crucial for maintaining the material quality of bone. Specifically, osteocytes are known to dynamically integrate biochemical and physical cues to modulate their signaling pathways, most notably the transforming growth factor-beta (TGFb) pathway, which is critical for regulating bone quality through the process of perilacunar remodeling (PLR). Disruptions to TGFb and PLR result in defects in bone quality, which in turn contribute to bone fracture and dental implant failure. However, the mechanism through which osteocytes sense physical and biochemical cues to control these functions remains unknown, representing a key gap in understanding the regulation of bone quality. Previous work identified that physical cues modulate TGFb pathway function, and that PLR produces marked changes in extracellular matrix stiffness, thereby altering the physical cues experienced by cells. Our preliminary data suggest that PLR outcomes are also mechanosensitive, suggesting a feedback loop incorporating TGFb, PLR, and physical cues. Furthermore, our recent work identified a tension-dependent spatial organization of TGFb receptors (TbRs) at integrin-rich, mechanosensing focal adhesions (FAs), representing an exciting potential link between osteocyte function and a well-studied pathway of cellular mechanosensation. Specifically, TbR Type II (TbRII) molecules were excluded from FAs, while TbR Type I (TbRI) colocalizes with integrins at FAs. As TGFb signaling depends on complex formation between TbRI and TbRII, this phenomenon may represent a key regulatory mechanism of TGFb signaling. Therefore, I propose to test the hypothesis that mechanosensitive TGFb signaling in osteocytes is mediated by distinct organization of TbRs through a mechanism dependent on force-sensing by integrins. In addition to evaluating the role of integrin force-sensing in this process, this proposal uses high-resolution molecular and imaging tools to dissect the diverse set of dynamic physical cues experienced by osteocytes. Specifically, Aim 1 employs TbRII mutants to identify the specific subdomains required for the organization and function of TbRs. Aim 2 tests the degree to which TbR organization responds to physical cues by subjecting osteocytes to fluid flow. Finally, Aim 3 utilizes recently-characterized magnetoplasmonic nanoparticles (MPNs), which apply tunable force to single molecules with high spatiotemporal resolution while allowing for simultaneous imaging, to probe the extent to which force applied to integrins affects TbR organization and TGFb signaling. The proposed studies will (i) elucidate the molecular mechanisms by which TGFb responds to physical cues to regulate osteocyte function, (ii) identify TbR organization as a potential mechanism through which TGFb signaling responds to physical cues and (iii) develop a novel method with which to study receptor dynamics in response to force in real-time. Given the critical role of osteocyte signaling in maintaining bone quality, the proposed work is an important step toward elucidating the mechanism for the regulation of bone quality, potentially paving the way to future therapeutic targets.